Salivary secretions maintain the health of the oral cavity. Building on our past studies of saliva formation and its alteration during pathology, we are developing novel approaches to treat salivary gland (SG) dysfunction primarily using principles of gene therapy and tissue engineering. During this reporting period we have made considerable progress in many facets of our work. Our studies are directed at fundamental questions necessary to move gene therapy into the clinic for phase-I trials in three areas: irradiation (IR)-induced salivary hypofunction, systemic single protein deficiency disorders (SSPDDs), and Sjogren?s syndrome (SS). The treatment of most head and neck cancer patients includes IR. SGs in the IR field suffer irreversible damage. Much of our effort has gone to prevent such damage, as well as to restore function to existing damaged glands. Over the last several years, we have examined the usefulness of the stable nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol) for radioprotection of the SGs and during this reporting period, we have studied its pharmacokinetic characteristics in a murine model. Tempol (137.5 or 275 mg/kg, 10 minutes prior to IR) significantly reduced IR-induced salivary hypofunction (~50 - 60%). Intravenous or subcutaneous administration was as protective as intraperitoneal administration, while intramuscular delivery was ineffective. Topical use of Tempol, either as a mouthwash or gel led to radioprotectiion. The results strongly suggest that Tempol may be useful to protect SGs in head and neck cancer patients undergoing radiotherapy. An important step in developing a clinical therapy is moving from a rodent to a large animal pre-clinical model. During this reporting period we have established the value of the miniature pig (minipig) parotid gland as a model for IR-induced salivary hypofunction. This year we began extending IR repair studies, reported last year using AdhAQP1 in this model. Those studies demonstrated that localized delivery of AdhAQP1 to IR-damaged salivary glands increases salivary secretion, without significant general adverse events, in a large animal model. We have now begun to test serotype 2 and 5 adeno-associated viral (AAV) vectors in this model to provide stable hAQP1 expression and,hopefully, stable restoration of fluid secretion. Our past studies have shown that SGs are a useful target site for treating SSPDDs, particularly when the transgene encodes a constitutive pathway secretory protein. The ability to control transgene expression is essential for clinical application. Previously, in a proof-of-concept study, we showed that the rapamycin-inducible transcriptional regulation system can regulate protein expression after adenoviral-mediated gene transfer to SGs. To evaluate the potential ability to utilize this regulatory system for long-term control of transgene expression in this tissue, we employed a ?third generation?, single AAV serotype 2 viral vector encoding human erythropoietin (hEPO) under the control of a rapamycin inducible promoter. The vector, rAAV-TF2.3-hEPO (10e10 particles/animal), was delivered to mouse SGs. No detectable increase in serum hEPO or hematocrit levels was observed in the absence of rapamycin administration. However, rapamycin induced elevation of serum hEPO levels, as well as concomitant hematocrit changes, that were dose-dependent, completely reversible and relatively stable over six months (longest time studied), with no appreciable change in rapamycin responsiveness. Unfortunately, regulated secretory pathway proteins, when delivered as transgenes to SGs, are secreted predominantly into saliva. This is not useful for those proteins whose therapeutic function is required systemically, e.g., human growth hormone (hGH). One strategy to improve the efficiency of hGH secretion into the bloodstream involves manipulation of existing sorting signals. We made several mutations in the C-terminus of the hGH cDNA and tested them. One biologically active mutant containing substitution of E174A and E186A, and with an included C-terminal extension showed, in vivo, a relative increase in the proportion of constitutive pathway secretion seen from rat SGs, with a significantly lower saliva vs. serum secretion ratio (p=0.03). Although this mutant is unlikely to be therapeutically beneficial, these results suggest that the final destination of a transgenic secretory protein may be controlled by re-engineering its? sorting determinants. We continue to use the female non-obese diabetic (NOD) mouse model of SS. Mice develop spontaneous autoimmune sialadenitis and loss of salivary flow. In the current period we used the NOD model and evaluated if transfer of the vasoactive intestinal peptide (VIP) cDNA could be useful in management of SS. We constructed a serotype 2 AAV vector encoding the human vasoactive intestinal peptide transgene (rAAV2hVIP) and, when administered prior to disease onset, observed that it led to significantly improved salivary flow and a reduction of submandibular gland cytokines IL-2, IL-10, IL-12 (p70) and TNF-alpha, and serum RANTES, compared to control vector, although no difference in focus scores was detected. In addition to our gene transfer studies, we have continued to make progress toward the development of an artificial SG, as described in previous annual reports. During the past year we have focused on establishing an autologous, polarized, non-human primate epithelial cell system to use in the device and thus facilitate in vivo functional testing. Using methods similar to those previously reported for human cells, we have achieved a reproducible means of obtaining these cells and we have begun to characterize their phenotype and function.